1. Field of the Invention
This invention relates to lyophilized aqueous parenteral solutions of physiologically active proteins and polypeptides attached to low diol polyalkylene oxide combined with the cryoprotectant cyclodextrin.
More particularly, this invention relates to a lyophilized aqueous parenteral solution of superoxide dismutase attached to low diol polyethylene glycol combined with the cryoprotectant cyclodextrin.
2. Reported Developments
Biologically active proteins, particularly enzymes and peptide hormones, have been long considered as ideal drugs for the treatment of various diseases due to their specificity and rapid catalytic action. Such enzymes include:
Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3; "uricase"); Hydrogen-peroxide: hydrogen-peroxide oxidoreductase (1.11.1.6; "catalase"); Cholesterol, reduced--NADP: oxygen oxidoreductase (20-.beta.-hydroxylating) (1.14.1.9; "Cholesterol 20-hydroxylase"). PA0 Transferases such as: UDP glucuronate glucuronyl-transferase (acceptor unspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose: .alpha.-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12). PA0 Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17; lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1; "Asparaginase"). PA0 Lyases such as: Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase (4.1.2.12; "aldolase"). PA0 Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase) and PA0 Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5). PA0 Insulin, ACTH, Glucagon, Somatostatin, Somatotropin, Thymosin, Parathyroid hormone, Pigmentary hormones, Somatomedin, Erythropoietin, Luteinizing hormone, Chorionic Gonadotropin, Hypothalmic releasing factors, Antidiuretic hormones, Thyroid stimulating hormone, Calcitonin and Prolactin. PA0 from about 150 to about 150,000 U/ml, preferably, PA0 from about 25,000 to about 150,000 U/ml, most preferably, PA0 from about 50,000 to about 150,000 U/ml of a covalently bound low diol polyethylene oxide/protein; PA0 from about 0.1 to about 20% w/v, preferably, PA0 from about 1.0 to about 15% w/v, and most preferably, PA0 from about 5 to about 10% w/v of cyclodextrin; and PA0 from about 0.01 to about 50 mM of a buffer at a pH of 5.7 to 6.5. PA0 Recombinant human interleukin-4 (rhuIL-4); PA0 Protease Subtilisin Carlsberg; PA0 Superoxide dismutases such as bovine, human, and various recombinant superoxide dismutases such as recombinant human superoxide dismutase (rhuSOD); PA0 Oxidoreductases such as: Urate: oxygen oxidoreductase (1.7.3.3; "uricase"); Hydrogen-peroxide: hydrogen-peroxide oxidoreductase (1.11.1.6; "catalase"); Cholesterol, reduced--NADP: oxygen oxidoreductase (20-.beta.-hydroxylating) (1.14.1.9; "Cholesterol 20-hydroxylase"); PA0 Transferases such as: UDP glucuronate glucuronyl-transferase (acceptor unspecific) (2.4.1.17; "UDP glucuronyltransferase"); UDP glucose: .alpha.-D-Galactose-1-phosphate uridylyltransferase 2.7.7.12); PA0 Hydrolases such as: Mucopeptide N-acetylmuramyl-hydrolase (3.2.1.17; lysozyme); Trypsin (3.4.4.4); L-Asparagine aminohydrolase (3.5.1.1; "Asparaginase"); PA0 Lyases such as: Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase (4.1.2.12; "aldolase"); PA0 Isomerases such as D-Xylose ketol-isomerase (5.3.1.5; xylose isomerase); and PA0 Ligases such as: L-Citrulline: L-aspartate ligase (AMP) (6.3.4.5). PA0 Insulin; ACTH; Glucagon; Somatostatin; Somatotropin; Thymosin; Parathyroid Hormone; Pigmentary Hormones; Somatomedin; Erythropoietin; Luteinizing Hormone; Chorionic Gonadotropin; Hypothalmic Releasing Factors; Antidiuretic Hormones; Thyroid Stimulating Hormone; Calcitonin; Prolactin; Interferons (alpha, beta and gamma); Antibodies (IgG, IgE, IgM, IgD); Interleukins 1, 2, 3, 4 and 7; Granulocyte Colony Stimulating Factor (GCSF); Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF); Tumor Necrosis Factor (TNF); Platelet Derived Growth Factor (PDGF); Epidermal Growth Factor (EGF); Nerve Growth Factor (NGF); Bone Growth Factor (BGF); Growth Hormone Releasing Factor (GHRF); Papain; Chymotrypsin; Thermolysin; Streptokinase and Activase.
The peptide hormones include:
Therapy with physiologically active proteinaceous substances, particularly with non-human enzymes, has been less than successful due in part to their relatively short half-lives and to their respective immunogenicities. Upon administration, the host defense system responds to remove the foreign enzymes by initiating the production of antibodies thereto, thereby substantially reducing or eliminating their therapeutic efficacies. Repeated administration of foreign and of otherwise short lived human enzymes is essentially ineffective, and can be dangerous because of concomitant allergic response. Various attempts have been taken to solve these problems, such as through microencapsulation, entrapment in liposomes, genetic engineering and attachment of the enzymes to polymers. Among the attempts the most promising appears to be the chemical attachment of the proteinaceous substances to polyalkylene oxide (PAO) polymers and particularly polyethylene glycols (PEG). The following illustrates these attempts.
U.S. Pat. No. 4,179,337 discloses the use of polyethylene glycol or polypropylene glycol coupled to proteins to provide a physiologically active non-immunogenic water soluble polypeptide composition in which the polyethylene glycol (hereinafter sometimes referred to as PEG) serves to protect the polypeptide from loss of activity without inducing substantial immunogenic response. The methods described in the patent for the coupling of polyethylene glycol to a protein involve either the conversion of a protein amino group into an amide or pseudoamide, with consequent loss of charge carrying capacity of the amino group, or the introduction at the amino group of the protein, or vicinal to it, of a heteroatom substituent such as a hydroxyl group or of a ring system that is not repeated in the polymer backbone.
Veronese, F. M., Boccu, E., Schaivon, O., Velo, G. P., Conforti, A., Franco, L., and Milanino, R., in Journal of Pharmacy and Pharmacology, 35, 757-758 (1983), reported that when bovine erythrocyte derived superoxide dismutase is modified with a polyethylene glycol carboxylic acid N-hydroxysuccinimide active ester, the half-life of the enzyme in rats is increased over that of the unmodified protein.
European Patent Application 0 200 467 of Anjinomoto, Inc. describes superoxide dismutase that is chemically modified by a polyalkylene oxide (PAO) which is functionalized at both ends of the polymer with activated carboxyl coupling groups, each capable of reacting with protein. Because the activated coupling sites are located at opposite ends of the polymer chain, it is unlikely that the presence of an activated group at one of the polymer can have a significant effect on the reactive nature of the group at the other end of the polymer. These polymers are capable of reacting at both ends to cross-couple with proteins to form copolymers between the protein and the polyalkylene oxide. Such copolymers do not have well defined or molecularly stoichiometric compositions.
Veronese, F. M. et al in Journal of Controlled Release, 10, 145-154 (1989) report that the derivatization with monomethoxypolyethylene glycol (hereinafter sometimes referred to as MPEG) of superoxide dismutase (hereinafter sometimes referred to as SOD) gives a heterogenous mixture of products. Heterogeneity was demonstrated to depend on the presence of bifunctional polyethylene glycol (DPEG) in the monofunctional methoxylated molecules.
These attempts, in general, have resulted in somewhat longer half-life and reduced immunogenicity of the proteinaceous physiologically active substances. However, it appears that further improvements are necessary to successfully treat a variety of diseases with these promising biological substances.
In co-pending patent application Ser. No. 07/936,416 (which is incorporated herein by reference) it is disclosed that biologically active proteinaceous substances can be made to possess longer half-life and less immunogenic properties by chemically modifying them using low diol polyalkylene oxide, preferably polyethylene glycol. The formulations disclosed have distinct advantages over the prior art disclosed formulations of polyethylene glycol-modified, proteinaceous substances.
During storage in the liquid state, polyethylene glycol proteinaceous molecules are hydrolyzed to a mixture of free polyethylene glycol, polyethylene glycol-protein and succinate-protein moieties. To prevent such a destabilization process, the formulations may be lyophilized. With lyophilization, however, the concentration of protein and stabilizers is at high levels and, depending on the excipients employed, deleteriously influence the degree of intermolecular aggregation that occurs during storage.
It has been found that cyclodextrins inhibit the rate of intermolecular aggregation of covalently attached low diol polyethylene glycol-proteins during their storage, and therefore, provide for extended shelf-life.
Cyclodextrins are known in the art to possess the ability to form inclusion complexes and to have concomitant solubilizing properties. Derivatives of cyclodextrins are also known to possess these properties. Their use is illustrated by the following patents.
U.S. Pat. No. 4,596,795, relates to the administration of sex hormones in the form of their complexes with hydrophilic derivatives of cyclodextrin, such as poly-.beta.-cyclodextrin and hydroxypropyl-.beta.-cyclodextrin, by sublingual or buccal routes. The complexes were found highly water-soluble and effective by comparison to other cyclodextrin derivatives.
U.S. Pat. No. 4,727,064 discloses pharmaceutical preparations consisting of a drug having low water solubility and an amorphous water-soluble cyclodextrin-based mixture. The addition of the cyclodextrin-based mixture improves the dissolution properties of the drug. The cyclodextrin-based mixture is prepared form .alpha., .beta.- or .gamma.-cyclodextrin which were rendered amorphous through non-selective alkylation.
International Application No. PCT/US89/04099 (WO 90/03784) describes a lyophilized composition comprising a polypeptide and a stabilizing/solubilizing amount of cyclodextrin selected from the group consisting of hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosyl derivatives of .beta.- and .gamma.-cyclodextrin.
U.S. Pat. No., 4,983,586 discloses a method for decreasing the incidence of precipitation of a lipophilic and/or water-labile drug occurring at the injection site, when the drug is being parenterally administered, comprising administering the drug in an aqueous solution containing about 20% to 50% hydroxypropyl-.beta.-cyclodextrin.
A large number of drugs are claimed including: antineoplastics, sedatives, tranquilizers, anticonvulsants, antidepressants, hypnotics, muscle relaxants, antisposmodics, anti-inflammatories, anticoagulants, cardiotonics, vasodilators and anti-arrhythmics.
We have surprisingly found that lyophilized parenteral formulations comprising conjugated low diol polyoxyethylene oxide and a physiologically active protein or polypeptide in a complex with cyclodextrin provide stability on extended shelf-life to the formulations without intramolecular aggregation.